Cloning and Characterization of Escherichia coli to Express Single-Chain Antibodies Against Clostridium difficile Toxins A and B

Clostridium difficile associated disease (CDAD) affects thousands of hospital patients every year. This disease is thought to result from overgrowth of C. difficile following disruption of the gut microflora after antibiotic treatment.  CDAD occurs because this Gram-positive microbe produces two cytotoxins, toxin A (TcdA) and toxin B (TcdB). The most worrisome aspects of the current C. difficile epidemic are the emergence of bacterial resistance to front-line antibiotics and the appearance of a more virulent strain of C. difficile.  The need for novel therapeutic and prophylactic approaches to CDAD is urgent. Using the paratransgenic strategy, we propose to deliver neutralizing single-chain antibodies (scFv’s) to TcdA and TcdB to gut mucosal surfaces via the probiotic, E. coli Nissle (EcN). In previous work, we had demonstrated that EcN persisted for up to two weeks in the gut of the CDAD mouse model. Here, we show the subcloning of the scFv’s against TcdA and TcdB into a new expression system.  These plasmids will be transformed into an optimized laboratory E. coli strain.  Clones harboring the TcdA and TcdB scFv containing plasmids will be characterized for growth and optimized for scFv expression.  Once protein expression is optimized, the scFv’s will be purified for biochemical characterization and in vitro functional assays.  These studies will then be repeated in EcN cells prior to further in vivo studies in the CDAD mouse models.  The successful expression of the scFv’s in the gut of these animals is expected to decrease the occurrence of CDAD.